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Probing the Antimicrobial Action of Polymyxin B1 and Melittin VIA Coarse-Grained Molecular Dynamics Simulations
28a
Sunday, February 28, 2016
Platform: Membrane Active Peptides and Toxins 153-Plat A Pore Model or the Carpet Model? The Mode of Action of AMPs on E. Coli Spheroplasts Yen Sun, Tzu-Lin Sun, Huey W. Huang. Physics and Astronomy, Rice University, Houston, TX, USA. One fundamental issue in the field of AMPs has been whether the action of AMPs produces pores in the membranes or disintegrates the membranes by the saturated surface binding of AMPs (the carpet model). In the absence of clear evidence, either mechanism seems to explain the phenomena of bacteria lysis. Even if the issue were resolved in the model membrane studies, it is not clear if the result of model membranes extends to bacterial membranes. Here we investigated the action of AMPs on E. coli spheroplasts. The absence of the outer membrane made it possible to observe the action of AMPs on the cytoplasmic membranes. Previously we found that the properties of the bacterial cell membranes are dominated by a membrane reservoir, significantly different from that of a GUV. Furthermore, these unique properties of bacterial membranes are metabolically maintained. We would like to know how the actions of AMPs on spheroplast membranes compare with the actions on GUVs. We compared the actions of human AMP LL37 and melittin on spheroplasts and GUVs. We developed a fluorescence recovery after photobleaching (FRAP) technique to examine the dye leakage through the bacterial membranes. AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the responses of GUVs. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of FRAP showed that the fluxes of dye molecules in and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. The effects of LL37 and melittin are qualitatively the same.
15N NMR). This simulation and a separate 2.5-ms trajectory of surfacebound peptides show large distortions of the bilayer/water interface (consistent with 31P NMR), which could be responsible for membrane disruption by piscidin. 156-Plat Directly Observing the Lipid-Dependent Self-Assembly and Pore Forming Mechanism of the Cytolytic Toxin Listeriolisyn O Estefania S. Mulvihill1, Katharina van Pee2, Stefania Mari1, ¨ zkan Yildiz2. Daniel J. Mueller1, O 1 BSSE, ETH, Basel, Switzerland, 2Department of Structural Biology, MaxPlanck-Institute of Biophysics, Frankfurt, Germany. Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild- type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit- shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.
154-Plat Probing the Antimicrobial Action of Polymyxin B1 and Melittin VIA Coarse-Grained Molecular Dynamics Simulations Damien F. Jefferies, Pin-Chia Hsu, Syma Khalid. Chemistry, University of Southampton, Southampton, United Kingdom. Many pathogenic Gram-negative bacteria are progressively acquiring resistance to the available spectrum of antibiotic drugs. Cationic antimicrobial peptides, which are known to exhibit rapid in vitro bactericidal activity, and a low propensity for resistance development, have been identified as promising candidates for the development of novel antibacterial agents. Rational development of therapeutics based on antimicrobial agents requires a molecular-level understanding of their mechanism of action. To contribute to this endeavour, we have studied two very different antimicrobial peptides, the cyclic lipopeptide, polymyxin B1 and the single helix peptide, melittin. We have performed a series of coarse-grained molecular dynamics simulations to explore how both peptides interact with the outer membrane of Gram-negative bacteria. The microsecond timescale simulations reveal very different patterns of behaviour of the two peptides and also show the very marked effect of having an asymmetric membrane. The latter observation is particularly crucial as this key feature of the in vivo bacterial outer membrane is often neglected in computational and experimental studies.
157-Plat Lipid-Protein Partnering during Pore Formation of Fragaceatoxin C Koldo Morante1, Jose M.M. Caaveiro1, Kouhei Tsumoto1,2. 1 Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan, 2Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Pore-forming toxins (PFT) are proteins that target cell membranes forming water-filled pores across the lipid bilayer. The steps leading to pore formation in PFT begin with membrane partitioning of the water-soluble monomers and are followed by the oligomerization and penetration of the protein subunits through the lipid bilayer. Reshaping of the protein into a final pore is governed by the interplay between the toxin and the physico-chemical landscape of the membrane. For example, in actinoporins (PFT from sea anemones) the presence of sphingomyelin is a strong contributor in making the toxin active. Additionally, when cholesterol is abundant, actinoporins make use of conserved residues to penetrate the membrane. Protein restructuring and lipid redistribution during the formation of the pore often lead to pores with distinct architectures. The pore formed by fragaceatoxin C (FraC), an actinoporin secreted by the sea anemone Actinia fragacea, has been traditionally described to be lined by both protein a-helices and lipid headgroups in a structure resembling a torus (toroidal model). However, recent data point to other alternatives where the protein contribution predominates. Herein, we show our recent findings involving FraC-membrane interaction and how these associate to build a pore mainly composed of protein but where lipids also play an important role.
155-Plat The Disruptive State of the Membrane Active Antimicrobial Peptide Piscidin 1 Investigated by Multi-mS All-Atom Simulations and Solid-State NMR: Surface Defects are Favored over Stable Pores B. Scott Perrin Jr.1, Riqiang Fu2, Myriam Cotten3, Richard W. Pastor1. 1 Laboratory of Computational Biology, NHLBI/NIH, Rockville, MD, USA, 2 National High Magnetic Field Laboratory, Tallahassee, FL, USA, 3 Department of Chemistry, Hamilton College, Clinton, NY, USA. Antimicrobial peptides (AMPs) that disrupt bacterial inner membranes are promising therapeutics against the growing number of antibiotic-resistant bacteria. The mechanism of membrane disruption by the AMP piscidin 1 was examined with multi-microsecond all-atom molecular dynamics simulations and solid-state NMR spectroscopy. A 14-ms control simulation of an archetype barrel-stave alamethicin pore validated the methodology. The primary simulation was initialized with 20 peptides in 4 barrel-stave pores in a fully hydrated POPC/POPG bilayer. The 4 pores relaxed to toroidal by 200 ns, only one pore containing 2 transmembrane helices remained at 20 ms, and none of the 18 peptides released to the surface reinserted to form pores. These results imply that the population of toroidal pores is <4% (consistent with
158-Plat Inhibition of RTX Toxin Activity by the Nuclear Stain, Draq5 Angela C. Brown1, Joshua Webb2. 1 Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA, 2Bioengineering, Lehigh University, Bethlehem, PA, USA. The repeats-in-toxin (RTX) family of proteins includes numerous toxins produced by Gram negative bacteria, including Bordetella pertussis (adenylate cyclase toxin), Escherichia coli (a-hemolysin), and Aggregatibacter actinomycetemcomitans (leukotoxin, LtxA). The mechanism by which LtxA recognizes and kills target cells involves multiple steps, including recognition of a b2 integrin receptor (LFA-1), binding to cholesterol/lipid rafts, and membrane bilayer destabilization. In a confocal imaging experiment, we found that LtxA rapidly becomes internalized in Jn.9 cells, and colocalizes with lysosomes. However, when the cells were pretreated with Draq5, a common nuclear stain, LtxA was found to be associated with the membrane, but not inside the cell. This inhibition of internalization was correlated with a decrease in the activity of the toxin. We hypothesized that upon crossing the plasma membrane, Draq5 alters the packing of the bilayer in a manner that inhibits the interaction of LtxA with the membrane, thus decreasing its activity. We
Sunday, February 28, 2016
Platform: Membrane Active Peptides and Toxins 153-Plat A Pore Model or the Carpet Model? The Mode of Action of AMPs on E. Coli Spheroplasts Yen Sun, Tzu-Lin Sun, Huey W. Huang. Physics and Astronomy, Rice University, Houston, TX, USA. One fundamental issue in the field of AMPs has been whether the action of AMPs produces pores in the membranes or disintegrates the membranes by the saturated surface binding of AMPs (the carpet model). In the absence of clear evidence, either mechanism seems to explain the phenomena of bacteria lysis. Even if the issue were resolved in the model membrane studies, it is not clear if the result of model membranes extends to bacterial membranes. Here we investigated the action of AMPs on E. coli spheroplasts. The absence of the outer membrane made it possible to observe the action of AMPs on the cytoplasmic membranes. Previously we found that the properties of the bacterial cell membranes are dominated by a membrane reservoir, significantly different from that of a GUV. Furthermore, these unique properties of bacterial membranes are metabolically maintained. We would like to know how the actions of AMPs on spheroplast membranes compare with the actions on GUVs. We compared the actions of human AMP LL37 and melittin on spheroplasts and GUVs. We developed a fluorescence recovery after photobleaching (FRAP) technique to examine the dye leakage through the bacterial membranes. AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the responses of GUVs. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of FRAP showed that the fluxes of dye molecules in and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. The effects of LL37 and melittin are qualitatively the same.
15N NMR). This simulation and a separate 2.5-ms trajectory of surfacebound peptides show large distortions of the bilayer/water interface (consistent with 31P NMR), which could be responsible for membrane disruption by piscidin. 156-Plat Directly Observing the Lipid-Dependent Self-Assembly and Pore Forming Mechanism of the Cytolytic Toxin Listeriolisyn O Estefania S. Mulvihill1, Katharina van Pee2, Stefania Mari1, ¨ zkan Yildiz2. Daniel J. Mueller1, O 1 BSSE, ETH, Basel, Switzerland, 2Department of Structural Biology, MaxPlanck-Institute of Biophysics, Frankfurt, Germany. Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild- type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit- shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.
154-Plat Probing the Antimicrobial Action of Polymyxin B1 and Melittin VIA Coarse-Grained Molecular Dynamics Simulations Damien F. Jefferies, Pin-Chia Hsu, Syma Khalid. Chemistry, University of Southampton, Southampton, United Kingdom. Many pathogenic Gram-negative bacteria are progressively acquiring resistance to the available spectrum of antibiotic drugs. Cationic antimicrobial peptides, which are known to exhibit rapid in vitro bactericidal activity, and a low propensity for resistance development, have been identified as promising candidates for the development of novel antibacterial agents. Rational development of therapeutics based on antimicrobial agents requires a molecular-level understanding of their mechanism of action. To contribute to this endeavour, we have studied two very different antimicrobial peptides, the cyclic lipopeptide, polymyxin B1 and the single helix peptide, melittin. We have performed a series of coarse-grained molecular dynamics simulations to explore how both peptides interact with the outer membrane of Gram-negative bacteria. The microsecond timescale simulations reveal very different patterns of behaviour of the two peptides and also show the very marked effect of having an asymmetric membrane. The latter observation is particularly crucial as this key feature of the in vivo bacterial outer membrane is often neglected in computational and experimental studies.
157-Plat Lipid-Protein Partnering during Pore Formation of Fragaceatoxin C Koldo Morante1, Jose M.M. Caaveiro1, Kouhei Tsumoto1,2. 1 Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan, 2Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Pore-forming toxins (PFT) are proteins that target cell membranes forming water-filled pores across the lipid bilayer. The steps leading to pore formation in PFT begin with membrane partitioning of the water-soluble monomers and are followed by the oligomerization and penetration of the protein subunits through the lipid bilayer. Reshaping of the protein into a final pore is governed by the interplay between the toxin and the physico-chemical landscape of the membrane. For example, in actinoporins (PFT from sea anemones) the presence of sphingomyelin is a strong contributor in making the toxin active. Additionally, when cholesterol is abundant, actinoporins make use of conserved residues to penetrate the membrane. Protein restructuring and lipid redistribution during the formation of the pore often lead to pores with distinct architectures. The pore formed by fragaceatoxin C (FraC), an actinoporin secreted by the sea anemone Actinia fragacea, has been traditionally described to be lined by both protein a-helices and lipid headgroups in a structure resembling a torus (toroidal model). However, recent data point to other alternatives where the protein contribution predominates. Herein, we show our recent findings involving FraC-membrane interaction and how these associate to build a pore mainly composed of protein but where lipids also play an important role.
155-Plat The Disruptive State of the Membrane Active Antimicrobial Peptide Piscidin 1 Investigated by Multi-mS All-Atom Simulations and Solid-State NMR: Surface Defects are Favored over Stable Pores B. Scott Perrin Jr.1, Riqiang Fu2, Myriam Cotten3, Richard W. Pastor1. 1 Laboratory of Computational Biology, NHLBI/NIH, Rockville, MD, USA, 2 National High Magnetic Field Laboratory, Tallahassee, FL, USA, 3 Department of Chemistry, Hamilton College, Clinton, NY, USA. Antimicrobial peptides (AMPs) that disrupt bacterial inner membranes are promising therapeutics against the growing number of antibiotic-resistant bacteria. The mechanism of membrane disruption by the AMP piscidin 1 was examined with multi-microsecond all-atom molecular dynamics simulations and solid-state NMR spectroscopy. A 14-ms control simulation of an archetype barrel-stave alamethicin pore validated the methodology. The primary simulation was initialized with 20 peptides in 4 barrel-stave pores in a fully hydrated POPC/POPG bilayer. The 4 pores relaxed to toroidal by 200 ns, only one pore containing 2 transmembrane helices remained at 20 ms, and none of the 18 peptides released to the surface reinserted to form pores. These results imply that the population of toroidal pores is <4% (consistent with
158-Plat Inhibition of RTX Toxin Activity by the Nuclear Stain, Draq5 Angela C. Brown1, Joshua Webb2. 1 Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA, 2Bioengineering, Lehigh University, Bethlehem, PA, USA. The repeats-in-toxin (RTX) family of proteins includes numerous toxins produced by Gram negative bacteria, including Bordetella pertussis (adenylate cyclase toxin), Escherichia coli (a-hemolysin), and Aggregatibacter actinomycetemcomitans (leukotoxin, LtxA). The mechanism by which LtxA recognizes and kills target cells involves multiple steps, including recognition of a b2 integrin receptor (LFA-1), binding to cholesterol/lipid rafts, and membrane bilayer destabilization. In a confocal imaging experiment, we found that LtxA rapidly becomes internalized in Jn.9 cells, and colocalizes with lysosomes. However, when the cells were pretreated with Draq5, a common nuclear stain, LtxA was found to be associated with the membrane, but not inside the cell. This inhibition of internalization was correlated with a decrease in the activity of the toxin. We hypothesized that upon crossing the plasma membrane, Draq5 alters the packing of the bilayer in a manner that inhibits the interaction of LtxA with the membrane, thus decreasing its activity. We